A bispecific antibody against infectious bursal disease virus vp2 and chicken cr2

Abstract

The present application relates to the prevention and treatment of chicken infectious bursal disease, and in particular to a bispecific antibody against infectious bursal disease virus VP2 and chicken CR2. The present application provides a bispecific antibody comprising a first binding domain and a second binding domain; the first binding domain can bind to chicken CR2; the second binding domain can bind to infectious bursal disease virus VP2. The bispecific antibody can not only bind to infectious bursal disease virus, but also target the membrane surface molecule chicken complement receptor 2 of B cells, and can be used for the preparation of targeted genetic engineering subunit vaccine, and lays a foundation for the development of targeted chicken infectious bursal disease subunit vaccine.

Description

Technical Field

[0001] This invention relates to the prevention and treatment of infectious bursal disease in chickens, specifically to a bispecific antibody against infectious bursal disease virus VP2 and chicken CR2. Background Technology

[0002] Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive infectious disease caused by infectious bursal disease virus (IBDV). Immunization is the primary method for controlling IBD. Subunit vaccines are widely used for IBD prevention, but their induced protective efficacy is still insufficient. Targeted genetically engineered subunit vaccines can target protective antigens to bind to immune cells, thereby enhancing their immunogenicity; therefore, the development of targeted genetically engineered subunit vaccines is of great significance.

[0003] Currently, disease control still primarily relies on conventional vaccines such as inactivated vaccines. IBD cystic virus tissue oil emulsion inactivated vaccines are used for pre-laying breeder chickens or chicks in epidemic areas, offering good protection. IBD cytotoxic inactivated vaccines differ in preparation methods from oil emulsion inactivated vaccines, but generally, their effectiveness is not as good as cystic virus tissue inactivated vaccines. Conventional inactivated vaccines have lower efficacy and require larger doses, resulting in higher costs. To reduce the number of immunizations, researchers have developed multivalent vaccines, such as ND-IBD bivalent inactivated vaccines and ND-IB-IBD trivalent inactivated vaccines. Currently, moderately virulent live vaccines include strains such as BJ836, NF8, K85, and 228E; low-virulence live vaccines use the low-virulence IBDV strain A80. Compared to low-virulence live vaccines, moderately virulent live vaccines can overcome higher levels of maternal antibody interference, but they also have problems such as virulence reversion and damage to the body. Due to the continuous emergence of new IBDV variants, traditional vaccines can no longer meet market demands.

[0004] Antibodies are the material basis for vaccine preparation. Compared to other antibodies, bispecific antibodies can directly bridge immune cells and target cells, enhancing the killing effect of immune cells. Furthermore, they can block dual signaling pathways by simultaneously binding to two antigens on tumor cells, thereby reducing tumor drug resistance. Because naturally occurring antibodies contain two heavy chain variable regions and two light chain variable regions, there may be up to 16 potential recombinants during production, leading to significant antibody diversity. One way to overcome this problem is to prepare individual antigen-binding fragments (Fabs) and link them via chemical coupling (such as oxidation) or physical linkage by adding adaptor proteins.

[0005] The VP2 protein is the main structural protein of infectious bursal disease virus (IBDV), accounting for approximately 51% of the total protein. It is the primary host-protective antigen of IBDV in chickens, exhibiting virulence specificity and playing a decisive role in pathogenicity. The neutralizing epitopes on the VP2 protein are serotype-specific. Furthermore, the VP2 protein is involved in inducing the production of virus-neutralizing antibodies, inducing apoptosis, and varying antigenic virulence.

[0006] Chicken complement receptor 2 (ChCR2) is a B cell-associated glycoprotein with a transmembrane region, primarily expressed on the surface of mature B cells and follicular dendritic cells. Its main function is to play a crucial regulatory role in B cell proliferation, differentiation, and memory.

[0007] The study of bispecific single-chain antibodies against infectious bursal disease virus VP2 and chicken CR2 will lay the foundation for the development of targeted genetically engineered subunit vaccines. Summary of the Invention

[0008] To meet the needs of the above-mentioned fields, the present invention provides a bispecific antibody comprising a first binding domain and a second binding domain; the first binding domain can bind to chicken CR2; and the second binding domain can bind to infectious bursal disease virus VP2.

[0009] In the bispecific antibody of the present invention, each binding domain comprises a variable region (VH region) from the antibody heavy chain, wherein the VH region of the first binding domain specifically binds to chicken CR2, and the VH region of the second binding domain specifically binds to infectious bursal disease virus VP2. Optionally, the two binding domains are connected by a short polypeptide spacer. A non-limiting example of a polypeptide spacer is Gly-Gly-Gly-Gly-Ser (GGGGS) and its repeating sequences. Each binding domain may additionally comprise a variable region (VL region) from the antibody light chain, and the VH and VL regions of the first and second binding domains are connected to each other by a polypeptide linker.

[0010] The corresponding heavy chain variable regions (VH regions) and the corresponding light chain variable regions (VL regions) can be arranged from the N-terminus to the C-terminus in the following order: VH(CR2)-VL(CR2)-VH(VP2)-VL(VP2), VL(CR2)-VH(CR2)-VL(VP2)-VH(VP2) or VH(VP2)-VL(VP2)-VH(CR2)-VL(CR2), VL(VP2)-VH(VP2)- VL(CR2)-VH(CR2); or can be arranged in the following order: VH(CR2)-VL(CR2)-VL(VP2)-VH(VP2), VL(CR2)-VH(CR2)-VL(VP2) or VL(VP2)-VH(VP2)-VL(CR2), VH(VP2)-VL(VP2)-VL(CR2), VH(VP2)-VL(VP2)-VH(CR2).

[0011] Linkers used to connect the corresponding heavy chain variable regions and light chain variable regions are typically flexible and protease resistant. Preferred linkers contain glycine and / or serine residues. Preferred linkers are peptide linkers containing the amino acid sequence GGGGS or SGGGG.

[0012] The amino acid sequence of the epitope tag can be introduced at any position in the amino acid sequence of the binding agent and presents a circular form in the encoded protein structure, or its N-terminus or C-terminus is fused to the binding domain. Preferably, the epitope tag is a C-terminus fused to the binding domain. The epitope tag can be any type of epitope tag that is functional under native and / or denaturing conditions, preferably a histidine tag, and most preferably a 6-histidine tag.

[0013] In some embodiments of the present invention, the first binding domain comprises a heavy chain variable region and a light chain variable region of the anti-chicken CR2 antibody; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2.

[0014] In some embodiments of the present invention, the second binding domain comprises a heavy chain variable region and a light chain variable region of an antibody against infectious bursal disease virus VP2; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 3; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4.

[0015] In some embodiments of the present invention, the amino acid sequence of the above-mentioned bispecific antibody is shown in SEQ ID NO: 5.

[0016] The gene encoding the above-mentioned bispecific antibody also falls within the scope of protection of this invention.

[0017] In some embodiments of the present invention, the nucleotide sequence of the above-mentioned gene is shown in SEQ ID NO: 6.

[0018] The recombinant nucleic acid encoding the above-mentioned bispecific antibody also falls within the scope of protection of this invention.

[0019] Recombinant vectors containing the above-mentioned recombinant nucleic acids also fall within the scope of protection of this invention.

[0020] The recombinant vector can be any known vector, including plasmid vectors, granular vectors, phage vectors, viral vectors, or artificial chromosome vectors. The recombinant vector includes cloning vectors or expression vectors. The expression vector can be a eukaryotic expression vector, such as pcDNA3 or pTT3; or a prokaryotic expression vector, such as pET-28a, pET-32a, or pCold TF.

[0021] Host cells containing the above-mentioned recombinant nucleic acid or the above-mentioned recombinant vector are also within the scope of protection of this invention.

[0022] The host cell can be a eukaryotic cell or a host bacterium. The eukaryotic cell can be HEK293 cells, CHO cells, etc. The host bacterium can be a clonal strain or an expression strain. The clonal strain can be *Escherichia coli* DH5α. The expression strain can be *Escherichia coli* BL21(DE3).

[0023] The present invention also provides a method for preparing the above-mentioned bispecific antibody, characterized in that it includes: introducing the gene encoding the bispecific antibody into an expression vector to obtain a recombinant vector; introducing the recombinant vector into a host bacterium to obtain a recombinant bacterium; culturing the recombinant bacterium and inducing protein expression.

[0024] In the above preparation method, the expression vector can be pET-28a, pET-32a, or pCold TF. The host bacterium can be Escherichia coli BL21(DE3).

[0025] The application of the above-mentioned bispecific antibodies in the preparation of targeted genetically engineered subunit vaccines for the prevention and treatment of infectious bursal disease in chickens is also within the scope of protection of this invention.

[0026] The present invention also provides a targeted genetically engineered subunit vaccine comprising the above-mentioned bispecific antibody.

[0027] Based on the preparation of monoclonal antibodies against infectious bursal disease virus VP2 and chicken CR2, this invention uses RT-PCR to obtain the light chain and heavy chain gene fragments of the antibodies from hybridoma cells that secrete the above monoclonal antibodies. After cloning and sequencing, specific sequences are selected and spliced ​​in a specific order to obtain a bispecific gene (called BiBe-CR2-VP2 gene).

[0028] This invention utilizes prokaryotic expression plasmids (pET-28a, pET-32a, or pCold TF) and an E. coli expression system to successfully obtain high-purity soluble BiBe-CR2-VP2 recombinant protein.

[0029] The CCK-8 cytotoxicity assay results showed that the minimum safe concentration of BiBe-CR2-VP2 recombinant protein for chicken embryo fibroblasts was 9 μM. Figure 12 Indirect immunofluorescence assays demonstrated that the BiBe-CR2-VP2 recombinant protein could specifically bind to IBDV in infected chicken embryo fibroblasts. Figure 13 Indirect ELISA results using IBDV as the coating antigen showed that the minimum protein concentration at which the BiTe-CR2-VP2 recombinant protein bound to IBDV was 562.8 nM. Figure 14 B). ELISA results on chicken lymphoma cells (DT40) immobilized on an ELISA plate showed that the BiTe-CR2-VP2 recombinant protein specifically bound to DT40 cells, with a minimum binding dose of 0.39 μg of recombinant protein per 10,000 cells. Figure 14 A) indicates that the BiTe-CR2-VP2 recombinant protein can target and bind to chicken CR2, a membrane surface molecule of B cells. Further evaluation of the immunomodulatory activity of the BiTe-CR2-VP2 recombinant protein using a chicken spleen lymphocyte proliferation assay showed that a concentration of 2.25 μM of the recombinant protein significantly promoted chicken spleen lymphocyte proliferation. Figure 15 ).

[0030] Therefore, the BiBe-CR2-VP2 recombinant protein prepared in this invention can not only bind to infectious bursal disease virus, but also target and bind to chicken complement receptor 2, a membrane surface molecule of B cells, thus possessing the biological activity of bispecific antibodies. It can be used for the preparation of targeted genetically engineered subunit vaccines, laying the foundation for the development of targeted chicken infectious bursal disease subunit vaccines. Attached Figure Description

[0031] Figure 1 Agarose gel electrophoresis images of PCR-amplified CR2-VL, CR2-VH, VP2-VL, and VP2-VH genes. M represents DL2000, 1 is the CR2-VL gene band, 2 is the CR2-VH gene band, 3 is the VP2-VL gene band, and 4 is the VP2-VH gene band.

[0032] Figure 2Construction and identification of the pET-32a-BiBe-CR2-VP2 recombinant plasmid. A: Double enzyme digestion results of pET-28a-BiBe-CR2-VP2 and pET-32a plasmids, where M is DL 15000, 1 is the pET-28a-BiBe-CR2-VP2 plasmid control, 2 is the BamHI and HindIII double enzyme digestion product of pET-28a-BiBe-CR2-VP2 plasmid, 3 is the pET-32a plasmid control, and 4 is the BamHI and HindIII double enzyme digestion product of pET-32a. B: Ligation results of BiBe-CR2-VP2 with the linearized pET-32a vector, where M is DL 5000, and 1 is the ligation product of BiBe-CR2-VP2 with the linearized pET-32a vector. C: Double enzyme digestion identification of pET-32a-BiBe-CR2-VP2 plasmid, where M is DL 5000, 1 is pET-32a plasmid control, 2 is BiBe-CR2-VP2 control, and 3 is the BamHI and HindIII double enzyme digestion product of pET-32a-BiBe-CR2-VP2 plasmid.

[0033] Figure 3 Construction and identification of the pCold TF-BiBe-CR2-VP2 recombinant plasmid. A: Double enzyme digestion results of the pCold TF plasmid, where M is DL 15000, 1 is the pCold TF plasmid control, and 2 is the BamHI and HindIII double enzyme digestion product of the pCold TF plasmid. B: Ligation results of BiBe-CR2-VP2 with the pCold TF linearized vector, where M is DL 2000, and 1 is the ligation product of BiBe-CR2-VP2 with the pCold TF linearized vector. C: Double enzyme digestion identification results of the pCold TF-BiBe-CR2-VP2 plasmid, where M is DL 5000, 1 is the BiBe-CR2-VP2 control, 2 is the pCold TF plasmid control, and 3 is the BamHI and HindIII double enzyme digestion product of the pCold TF-BiBe-CR2-VP2 plasmid.

[0034] Figure 4 Expression results of recombinant BiBe-CR2-VP2 protein using pET-28a as a vector. Where M is the molecular weight standard of protein, 1 is the lysis buffer of the pET-28a empty vector control, 2 is the lysis buffer of the uninduced control, and 3 is the lysis buffer of BL21(DE3) induced culture containing pET-28a-BiBe-CR2-VP2.

[0035] Figure 5Purification results of BiBe-CR2-VP2 recombinant protein expressed by pET-28a vector. Where M represents the molecular weight of the protein, 1 is the supernatant of the lysate of BL21(DE3) induced culture containing pET-28a-BiBe-CR2-VP2 (before column chromatography), 2 is the breakthrough buffer, 3 is 20 mM imidazole elution buffer, 4 is 50 mM imidazole elution buffer, 5 is 100 mM imidazole elution buffer, 6 is 200 mM imidazole elution buffer, and 7 is 300 mM imidazole elution buffer.

[0036] Figure 6 Western blot identification results of recombinant BiBe-CR2-VP2 protein expressed by pET-28a vector. Where M represents the molecular weight standard of the protein, 1 is the lysate of the uninduced control, 2 is the supernatant of the lysate supernatant of BL21(DE3) induced culture containing pET-28a-BiBe-CR2-VP2, and 3 is the precipitate of the lysate supernatant of BL21(DE3) induced culture containing pET-28a-BiBe-CR2-VP2.

[0037] Figure 7 Expression and purification results of BiBe-CR2-VP2 recombinant protein using pET-32a as vector. Where M represents the molecular weight of the protein, 1 is the lysis buffer of the pET-32a empty vector control, 2 is the lysis buffer of the uninduced control, 3 is the lysis buffer of BL21(DE3) induced culture containing pET-32a-BiBe-CR2-VP2, 4 is the supernatant of the lysis buffer of BL21(DE3) induced culture containing pET-32a-BiBe-CR2-VP2 (before column chromatography), 5 is breakthrough buffer 1, 6 is breakthrough buffer 2, 7 is 20 mM imidazole elution buffer, 8 is 50 mM imidazole elution buffer, and 9 is 100 mM imidazole elution buffer.

[0038] Figure 8 Western blot identification results of recombinant BiBe-CR2-VP2 protein expressed by pET-32a vector. Where M is the molecular weight standard of protein, 1 is the lysate of the uninduced control, and 2 is the supernatant of the lysate supernatant of BL21(DE3) induced culture containing pET-32a-BiBe-CR2-VP2.

[0039] Figure 9Expression results of recombinant BiBe-CR2-VP2 protein using pCold TF as a vector. Where M represents the molecular weight of the protein, 1 is the lysis buffer of the uninduced control, 2 is the lysis buffer of the pCold TF empty vector control, 3 is the lysis buffer of BL21(DE3) induced culture containing pCold TF-BiBe-CR2-VP2, 4 is the supernatant of the lysis buffer of BL21(DE3) induced culture containing pCold TF-BiBe-CR2-VP2, and 5 is the precipitate of the lysis buffer of BL21(DE3) induced culture containing pCold TF-BiBe-CR2-VP2.

[0040] Figure 10 Purification results of recombinant BiBe-CR2-VP2 protein expressed by pCold TF vector. Where M represents the molecular weight standard of the protein, 1 is the lysis buffer of the uninduced control, 2 is the lysis buffer of BL21(DE3) induced culture containing pCold TF-BiBe-CR2-VP2, 3 is the supernatant of the lysis buffer of BL21(DE3) induced culture containing pCold TF-BiBe-CR2-VP2, 4 is the flow-through buffer, 5 is 50 mM imidazole elution buffer, 6 is 80 mM imidazole elution buffer, 7 is 100 mM imidazole elution buffer, 8 is 200 mM imidazole elution buffer, and 9 is 300 mM imidazole elution buffer.

[0041] Figure 11 Western blot identification results of recombinant BiBe-CR2-VP2 protein expressed by pCold TF vector. Where M is the molecular weight standard of protein, and l is the supernatant of lysate supernatant of BL2I(DE3) induced culture containing pCold TF-BiBe-CR2-VP2.

[0042] Figure 12 Effect of BiBe-CR2-VP2 recombinant protein on chicken embryo fibroblast activity. The x-axis represents the dilution ratio of BiBe-CR2-VP2 recombinant protein (1 mg / mL), and the y-axis represents chicken embryo fibroblast activity.

[0043] Figure 13 Indirect immunofluorescence was used to verify the interaction between the BiBe-CR2-VP2 recombinant protein and IBDV. pCold TF-BiBe-CR2-VP2 represents the BiBe-CR2-VP2 recombinant protein expressed via the pCold TF vector. The microscope magnification was 40×.

[0044] Figure 14ELISA was used to verify the interaction between the BiBe-CR2-VP2 recombinant protein and chicken lymphoma cells (DT40) and IBDV. A: ELISA results of the interaction between the BiBe-CR2-VP2 recombinant protein and DT40 cells. B: ELISA results of the interaction between the BiBe-CR2-VP2 recombinant protein and IBDV.

[0045] Figure 15 Effect of BiBe-CR2-VP2 recombinant protein on the proliferation of chicken spleen lymphocytes. The concentration values ​​in the figure are the concentration values ​​of BiBe-CR2-VP2 recombinant protein; the vertical axis represents the survival rate of chicken spleen lymphocytes. Detailed Implementation

[0046] The present invention will be described in detail below with reference to the embodiments. It should be understood that the following embodiments are only for explanation and illustration of the present invention and do not limit the scope of the present invention in any way.

[0047] Cells: The hybridoma cells (216D VP2 hybridoma cells) secreting monoclonal antibodies against infectious bursal disease virus VP2 and the hybridoma cells (4E5G CR2 hybridoma cells) secreting monoclonal antibodies against chicken complement receptor 2 used in the following examples were prepared in our laboratory and stored at the Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agricultural and Forestry Sciences. Transt1-T1 competent cells were purchased from Beijing TransGen Biotech Co., Ltd. Escherichia coli DH5α competent cells, Escherichia coli BL21(DE3) competent cells, chicken embryonic fibroblasts (DF-1), and chicken lymphoma cells (DT40) were provided by the Animal Disease Research Center of Beijing Academy of Agricultural and Forestry Sciences and are commercially available.

[0048] Virus: The infectious bursal disease virus (IBDV) used in the following examples was provided by the Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agricultural and Forestry Sciences. It is the known IBDV strain BJQ902, which has been published in the literature “Zhang Zhenhua, Li Lin, Jing Xiaodong, Zhang Jianwei, Shen Jia, Shi Aihua, Zheng Xiaolan, Huang Fengjun, Jiang Beiyu. Study on the propagation process of chicken infectious bursal disease virus strain BJQ902 on DF-1 cell line. Chinese Journal of Animal Husbandry and Veterinary Medicine. 2016, 43(10): 2775-2779.” The public can obtain this virus from the Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agricultural and Forestry Sciences.

[0049] Animals: SPF chickens were purchased from Beijing Merial Viton Laboratory Animal Technology Co., Ltd.

[0050] Vectors: pET-28a vector was provided by Beijing Branch of Sangon Biotech (Shanghai) Co., Ltd. pET-32a and pCold TF vectors were provided by the Animal Disease Research Center of Beijing Academy of Agricultural and Forestry Sciences. All the above vectors are commercially available products.

[0051] Reagents: TRNzol RNA extraction kit was purchased from Thermo Fisher Scientific, catalog number 15596026. FastQuant RT Super Mix FastQuant cDNA first-strand synthesis premix reagent was purchased from Tiangen Biotech (Beijing) Co., Ltd., catalog number KR108. pEASY-Blunt Simple cloning Vector was purchased from TransGen Biotech Co., Ltd., catalog number CB111-01. PFU polymerase and 10×PFU Buffer were purchased from Zhongke Ruitai (Beijing) Biotechnology Co., Ltd. BamH I enzyme and Hind III enzyme were purchased from NEB. Mouse anti-His-Tag mAb was purchased from ABclonal, catalog number AE003. Anti-Mouse IgG-FITC was purchased from SIGMA, catalog number F0257. CCK-8Cell Counting Kit was purchased from Novizan, catalog number A311-01. The HRP-labeled His antibody was purchased from Kangwei Century Biotechnology Co., Ltd., catalog number CW0285. The 2×T5 Mix was purchased from Qingke Biotechnology Co., Ltd., catalog number TSE005.

[0052] Unless otherwise specified, the reagents used in the following examples are all conventional reagents in the art, commercially available or prepared according to conventional methods in the art, and are of laboratory purity. Unless otherwise specified, the experimental methods and conditions used in the following examples are conventional experimental methods and conditions in the art, and can be found in relevant experimental manuals, public literature, or manufacturer's instructions. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0053] Example 1. Obtaining bispecific antibodies against infectious bursal disease virus VP2 and chicken CR2

[0054] 1. Obtaining bispecific antibody genes

[0055] The inventors previously obtained hybridoma cells secreting monoclonal antibodies against infectious bursal disease virus VP2 (referred to as 216D VP2 hybridoma cells) and hybridoma cells secreting monoclonal antibodies against chicken complement receptor 2 (referred to as 4E5G CR2 hybridoma cells). Total RNA was extracted from 216D VP2 and 4E5G CR2 hybridoma cells using the TRNzol RNA extraction kit (Thermo Fisher, 15596026). First-strand cDNA was synthesized from hybridoma cells using FastQuant RT Super Mix FastQuant cDNA first-strand synthesis premix reagent (Tiangen, KR108). Using the cDNA as a template, the VH and VL genes of the monoclonal antibodies were amplified by PCR.

[0056] The primers for amplifying the VH gene (VP2-VH gene) of VP2 monoclonal antibody are VH-F and VP2-VH-R. The primers for amplifying the VL gene (VP2-VL gene) of VP2 monoclonal antibody are VL-F and VL-R. The primers for amplifying the VH gene (CR2-VH gene) of CR2 monoclonal antibody are VH-F and CR2-VH-R. The primers for amplifying the VL gene (CR2-VL gene) of CR2 monoclonal antibody are VL-F and VL-R. The nucleotide sequences of the primers are as follows:

[0057] VH-F: 5'-SARGTNMAGCTSGSAGSAGTC-3'

[0058] VP2-VH-R: 5'-CTTGACCAGGCATCCTAGAGTCA-3'

[0059] CR2-VH-R: 5'-AGGGGCCAGTGGATAGACTGATGG-3'

[0060] VL-F: 5'-GAYATTGTGMTSACCMCARWCTMCA-3'

[0061] VL-R: 5'-GGATACAGTTGGTGCAGCATC-3'

[0062] In the nucleotide sequences of the primers above, S = C / G, R = A / G, N = A / C / G / T, M = A / C, Y = C / T, and W = A / T.

[0063] The PCR reaction system consisted of: 10×pfu buffer, 5 μL; forward primer (10 μmol / μL), 2 μL; reverse primer (10 μmol / μL), 2 μL; template cDNA, 4 μL; dNTP mixture (2.5 nmol / L), 4 μL; pfu polymerase, 1 μL; and ddH2O, 32 μL.

[0064] The PCR reaction program was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, Tm annealing for 35 s, 72℃ extension for 1 min, repeated for 30 cycles; 72℃ extension for 5 min. The annealing temperature (Tm) for amplifying the VL gene of the VP2 monoclonal antibody was 50℃, and the annealing temperature (Tm) for amplifying the VH gene of the VP2 monoclonal antibody, as well as the VH and VL genes of the CR2 monoclonal antibody, was 55℃.

[0065] The PCR products were identified by 1% agarose gel electrophoresis. The results are as follows: Figure 1 As shown, the obtained CR2-VL gene band (lane 1) is 360bp, the CR2-VH gene band (lane 2) is 387bp, the VP2-VL gene band (lane 3) is 356bp, and the VP2-VH gene band (lane 4) is 448bp, which are consistent with the expected size.

[0066] The PCR products were excised from the gel and ligated into a pEASY-Blunt Simple cloning vector (TransGold, CB111-01). The ligation reaction mixture was: PCR product, 1 μL; pEASY-Blunt Simple cloning vector, 2 μL; ddH2O to a final volume of 10 μL. The mixture was gently mixed and incubated at room temperature for 5 minutes. After the reaction, the centrifuge tubes were placed on ice.

[0067] Transformation: Add 10 μL of the ligation product to Trans1-T1 competent cells (Full-Gold), gently swirl to mix, and incubate on ice for 30 minutes. Heat shock at 42°C for 30 seconds, then immediately place on ice for 2 minutes. Add 250 μL of LB medium equilibrated to room temperature and incubate at 37°C for 1 hour at 200 rpm. Mix 8 μL of 500 mM IPTG and 40 μL of 20 mg / mL X-gal, spread evenly on LB plates, and incubate at 37°C for 30 minutes. After the IPTG and X-gal have been absorbed, spread 200 μL of the bacterial culture evenly on a plate and incubate overnight at 37°C. Pick white single clones and add them to 10 μL of sterile water, vortex to mix, and add 1 μL of the bacterial culture to a 25 μL PCR system. Identify positive clones using the M13 forward and reverse primers. Send clones identified positive by bacterial culture PCR to Sino-American Taihe Biotechnology Co., Ltd. for sequencing identification. The correct sequences were extracted and identified. Specific sequences were selected and assembled in a specific order. Then, the Beijing branch of Sangon Biotech (Shanghai) Co., Ltd. was commissioned to optimize the assembled sequences and synthesize the whole genome. The encoding gene for bispecific antibodies against infectious bursal disease virus VP2 and chicken complement receptor 2 was obtained and named BiBe-CR2-VP2 gene.

[0068] Full-length sequence of the BiBe-CR2-VP2 gene:

[0069]

[0070] 2. Construction of expression carriers

[0071] (1) Construction of pET-28a-BiBe-CR2-VP2 recombinant plasmid

[0072] BiBe-CR2-VP2 was ligated into the pET-28a plasmid by Sangon Biotech (Shanghai) Co., Ltd., Beijing Branch. The pET-28a-BiBe-CR2-VP2 plasmid was transformed into DH5α competent cells. 100 μL of the recovered bacterial culture was plated on Kana / LB solid medium and incubated overnight at 37°C. PCR identification was performed using specific primers BiBe-F and His-BiBe-R. The nucleotide sequences of the primers are as follows:

[0073] BiBe-F: 5'-GGATCCATGGATATTGTGCTGACCCAGACC-3'

[0074] His-RiBE-R: 5'-CCCAAGCTTTTAATGATGATGATGATGATGATTAGGGCAG-3'

[0075] The PCR system consisted of: 10 μL of 2×T5 Mix, 1 μL of BiBe-F, 1 μL of His-BIBe-R, and 1 μL of bacterial culture, with ddH2O added to a final volume of 20 μL. The PCR program was: 98℃ for 2 min; 98℃ for 10 s, 55℃ for 10 s, 72℃ for 10 s (35 cycles); 72℃ for 2 min. Plasmids from single colonies that tested positive by PCR were extracted and transformed into BL21(DE3) competent cells. Single colonies were picked and sequenced to identify BL21(DE3) cells containing the correct pET-28a-BiBe-CR2-VP2 sequence.

[0076] (2) Construction of pET-32a-BiBe-CR2-VP2 recombinant plasmid

[0077] The pET-28a-BiBe-CR2-VP2 and pET-32a plasmids were double-digested using BamHI and HindIII restriction endonucleases, respectively. The digestion system consisted of: 2.1 μL Neb Buffer, 1 μL BamHI enzyme, 1 μL HindIII enzyme, 1 μg plasmid, and ddH2O to a final volume of 20 μL. Digestion was carried out at 37°C for 3 h. After electrophoresis of the digestion products, the BiBe-CR2-VP2 gene fragment and the pET-32a linearized vector were recovered from the gel and then ligated. The ligation system consisted of: 0.3 pmol pET-32a linearized vector, 0.9 pmol BiBe-CR2-VP2 gene fragment, 2 μL T4 DNA ligase, 2 μL T4 DNA ligase buffer, and ddH2O to a final volume of 20 μL. Ligation was performed overnight at 16°C. All ligation products were transformed into DH5α competent cells, and bacterial colony PCR was performed using specific primers BiBe-F and His-BiBe-R for identification. Plasmids from clones that showed positive PCR results were extracted and double-digested with BamHI and HindIII restriction endonucleases for identification. Figure 2 The plasmids that were correctly identified by enzyme digestion were sequenced. BL21(DE3) competent cells were transformed with the recombinant plasmids that had the correct sequence, and bacterial culture PCR was performed using specific primers BiBe-F and His-BiBe-R to identify BL21(DE3) cells containing pET-32a-BiBe-CR2-VP2.

[0078] (3) Construction of pCold TF-BiBe-CR2-VP2 recombinant plasmid

[0079] pCold TF was double-digested with BamHI and HindIII restriction endonucleases, using the same digestion system and conditions as described in (2). The digestion products were electrophoresed and the pCold TF linearized vector was recovered via gel extraction. The pCold TF linearized vector was ligated with the BiBe-CR2-VP2 gene fragment obtained in (2), using the same ligation system as described in (2). The ligation product was transformed into DH5α competent cells, and 100 μL of the recovered bacterial culture was plated on Amp / LB solid medium and incubated overnight at 37°C. PCR identification of the bacterial culture was performed using specific primers BiBe-F and His-BiBe-R. Plasmids from clones that showed positive PCR results were extracted and double-digested with BamHI and HindIII restriction endonucleases for identification. Figure 3 The plasmids that were correctly identified by enzyme digestion were sequenced. BL21(DE3) competent cells were transformed with the recombinant plasmids that had the correct sequences, and BL21(DE3) cells containing pCold TF-BiBe-CR2-VP2 were identified by colony PCR using primers BiBe-F and His-BiBe-R.

[0080] 3. Expression and purification of recombinant proteins

[0081] The BL21(DE3) expression strains obtained in step 2 above were inoculated into LB liquid medium and cultured overnight at 37°C with shaking at 180 rpm. The overnight culture was then inoculated into LB liquid medium at a 1:100 volume ratio and cultured at 37°C with shaking at 180 rpm. When the OD600 reached 0.6, IPTG was added to a final concentration of 0.6 mM, and the culture was induced at 25°C with shaking at 180 rpm for 8–20 h. Simultaneously, BL21(DE3) containing pET-28a-BiBe-CR2-VP2, BL21(DE3) containing pET-32a-BiBe-CR2-VP2, and BL21(DE3) containing pCold TF-BiBe-CR2-VP2 were induced to express their contents. BL21(DE3) containing the corresponding recombinant plasmids that were not induced to express their contents served as an uninduced control. BL21(DE3) containing the corresponding empty vectors was induced to express their contents under the same conditions, serving as an empty vector control.

[0082] Collect the bacterial culture that induces protein expression, centrifuge to collect the bacterial cell pellet, wash three times with PBS buffer, resuspend, sonicate, centrifuge, add 5× protein loading buffer to the supernatant and pellet respectively, boil for 10 min, and then perform 10% SDS-PAGE gel electrophoresis. Stain the SDS-polyacrylamide gel with Coomassie Brilliant Blue for 30 min, destain with destaining solution until the background is clear, and observe the results.

[0083] A large amount of bacterial culture inducing protein expression was collected, centrifuged to collect the bacterial cell pellet, resuspended in PBS buffer, sonicated, and centrifuged again to collect the supernatant. The supernatant was filtered through 0.2 μm filter paper and subjected to nickel agarose affinity chromatography. The eluents at 100 mM and 200 mM imidazole concentrations were collected and subjected to 12% SDS-PAGE gel electrophoresis. For Western blot identification, the eluent was subjected to 10% SDS-PAGE gel electrophoresis and then transferred to a PVDF membrane. The membrane was blocked with 5% skim milk and immunoblot was performed using HRP-labeled His antibody (Kangwei Century, CW0285) diluted 1:1000. The immunoblot was then developed using an ECL chemiluminescence kit.

[0084] The expression results of the BiBe-CR2-VP2 recombinant protein using pET-28a as a vector are as follows: Figure 4 As shown, the protein size is approximately 60 kDa, consistent with the predicted result. Elution was performed using 20 mM imidazole, 50 mM imidazole, 100 mM imidazole, 200 mM imidazole, and 300 mM imidazole, respectively. Impurities were washed away at 20 mM and 50 mM, and the BiBe-CR2-VP2 recombinant protein eluted at 100 mM, but not as a single band. Figure 5 After 8 hours of IPTG induction culture, the bacterial culture was lysed by sonication. Western blot analysis was performed on the supernatant and precipitate (inclusion bodies) of the lysate containing pET-28a-BiBe-CR2-VP2 from BL21(DE3) induction culture using HRP-labeled His antibody (Kangwei Century, CW0285). The results showed that the recombinant BiBe-CR2-VP2 protein was correctly expressed in both the supernatant and inclusion bodies, but the protein expression level in the inclusion bodies was significantly higher than that in the supernatant. Figure 6 ).

[0085] The expression results of recombinant BiBe-CR2-VP2 using pET-32a as a vector are as follows: Figure 7 As shown, the protein size is approximately 72 kDa, consistent with the predicted result. During purification, impurities were washed away at 20 mM and 50 mM, and the BiBe-CR2-VP2 recombinant protein eluted at 100 mM, but not as a single band. The bacterial culture induced by IPTG for 8 hours was lysed by sonication, and the supernatant of the lysate was subjected to Western blot analysis using an HRP-labeled His antibody (Kangwei Century, CW0285) to identify the recombinant protein. Figure 8 ).

[0086] The expression results of recombinant BiBe-CR2-VP2 using pCold TF as a vector are as follows: Figure 9As shown, the protein size is approximately 110 kDa, consistent with the predicted result. The bacterial culture induced by IPTG for 20 h was subjected to ultrasonic lysis, and the supernatant was used for protein purification. The results showed that the BiBe-CR2-VP2 recombinant protein was eluted at imidazole concentrations of 200 mM and 300 mM. Figure 10 The bacterial culture induced by IPTG for 8 hours was lysed by sonication. The supernatant of the lysate was subjected to Western blot analysis using an HRP-labeled His antibody (Kangwei Century, CW0285) to identify the recombinant protein. Figure 11 ).

[0087] Example 2. Identification of the bioactivity of bispecific antibodies against infectious bursal disease virus VP2 and chicken CR2.

[0088] The BiTe-CR2-VP2 recombinant protein used in the following experiments was the BiTe-CR2-VP2 recombinant protein expressed and purified by the pCold TF vector in Example 1.

[0089] 1. CCK-8 cytotoxicity assay

[0090] Chicken embryo fibroblast cell line (DF-1) was used at 5×10 4 Each hole is laid in a 96-well plate for later use, with 5 × 10⁻⁶ holes. 5 Cells were seeded per well into 12-well plates containing cell spreaders. BiTe-CR2-VP2 recombinant protein (1 mg / mL) was serially diluted from 1:2 to 1:8112 and added to 100 μL per well of 96-well plates containing DF-1 cells. Four replicates were performed for each dilution. Blank and cell control wells were also included. The plates were incubated at 37°C for 4 h. After 4 h, 10 μL of CCK-8 solution (Novazia) was added to each well, and the plates were incubated at 37°C for 2 h. OD450 was then measured using a multi-well microplate reader, and cell viability and death rates were calculated. Results are as follows: Figure 12 As shown, the minimum safe concentration of BiBe-CR2-VP2 protein for chicken embryo fibroblasts is 9 μM (1:64 dilution).

[0091] 2. Indirect immunofluorescence assay

[0092] A 12-well plate filled with DF-1 cells was used. Each well was added with IBDV virus solution containing 100 TCID50. The cells were incubated at 37°C until approximately 70% cytotoxicity was achieved. Cells were then fixed with 4% paraformaldehyde for 1 hour, permeabilized with 0.1% Triton X-100 for 2 hours, and blocked with blocking buffer (0.5% BSA) at 37°C for 1 hour. Purified BiTe-CR2-VP2 recombinant protein (1 mg / mL) was diluted 1:64 with blocking buffer as the primary antibody. Mouse anti-His-Tag mAb (ABclonal, AE003) diluted 1:200 was used as the secondary antibody, and Anti-Mouse IgG-FITC (SIGMA, F0257) diluted 1:400 was used as the triple antibody for the immunoreaction. Finally, the nuclei were stained with DAPI. The cells were observed under a fluorescence microscope. A control group of CEF cells without virus infection was set up; a positive control group used 216D VP2 monoclonal antibody diluted 1:200 as the primary antibody; and a negative control group used SPF chicken negative serum as the primary antibody. IBDV virus control wells were also included. The results showed that BiBe-CR2-VP2 protein can specifically bind to IBDV infected in chicken embryo fibroblasts. Figure 13 ).

[0093] 3. Binding of BiTe-CR2-VP2 recombinant protein to chicken lymphoma cells (DT40)

[0094] DT40 cells were seeded onto ELISA plates and incubated at 37°C for 1 hour, followed by overnight incubation at 4°C. Cells were fixed with 4% cell tissue fixative and blocked with 5% skim milk, then incubated at 37°C for 2 hours. BiTe-CR2-VP2 recombinant protein (1 mg / mL) stock solution was serially diluted and added sequentially to ELISA plates, 100 μL per well, with 8 replicates per concentration, and incubated at 37°C for 1 hour. HRP-labeled His antibody (Kangwei Century, CW0285) was diluted 1:1000 using PBST and incubated at 37°C for 1 hour. TMB single-component chromogenic buffer (Solarbio) was added to each well, and the plates were incubated at 37°C for 15 minutes. The reaction was terminated by adding 2M sulfuric acid, and OD450 was detected using a multi-mode microplate reader. A P / N ratio greater than 2.1 was considered positive. Results are as follows. Figure 14 As shown in (A), the BiTe-CR2-VP2 recombinant protein can specifically bind to DT40 cells, with a minimum binding dose of 0.39 μg protein per 10,000 cells (the recombinant protein is diluted 1:256).

[0095] 4. Binding of BiTe-CR2-VP2 recombinant protein to IBDV

[0096] IBDV was diluted 10-fold with PBS starting at 1:10 on the ELISA plate, and then diluted to 1:10.7 The last row was treated with PBS only, incubated at 37°C for 1 hour, and then overnight at 4°C. Fixation was performed using 5% skim milk, incubated at 37°C for 2 hours. BiTe-CR2-VP2 recombinant protein stock solution (1 mg / mL) was serially diluted, one dilution per column, and added sequentially to the ELISA plate, incubated at 37°C for 1 hour. HRP-labeled His antibody (Kangwei Century, CW0285) was diluted 1:1000 using PBST, and incubated at 37°C for 1 hour. TMB single-component chromogenic buffer (Solarbio) was added to each well, incubated at 37°C for 15 minutes, and the reaction was stopped by adding 2M sulfuric acid. OD450 was detected using a multi-mode microplate reader. A P / N ratio greater than 2.1 was considered positive. Results are as follows: Figure 14 As shown in (B), the lowest protein concentration at which BiBe-CR2-VP2 recombinant protein binds to IBDV is 562.8 nM (the recombinant protein is diluted by a factor of 1:1024).

[0097] 5. CCK-8 cell proliferation assay

[0098] Chicken spleen lymphocytes were prepared and seeded into 96-well plates, 5 × 10⁶ cells / well. 5 Cells / well. 9 μM, 4.5 μM, 2.25 μM, and 1.125 μM BiTe-CR2-VP2 recombinant protein were added to 96-well plates containing chicken spleen lymphocytes, 100 μL per well, with 6 replicates per well. A blank control group and a cell control group were also included, with 6 wells each, for a total of 3 plates. After incubation at 37°C for 12 h, 10 μL of CCK-8 solution (Novizan) was added to each well for an additional 2 h of incubation. OD450 was measured using a multi-mode microplate reader, and cell viability and death rates were calculated. Results showed that a BiTe-CR2-VP2 protein concentration of 2.25 μM significantly promoted the proliferation of chicken spleen lymphocytes. Figure 15 ).

Claims

1. A bispecific antibody against infectious bursal disease virus VP2 and chicken CR2, the amino acid sequence of which is shown in SEQ ID NO:

5.

2. A gene encoding the bispecific antibody of claim 1, the nucleotide sequence of which is shown in SEQ ID NO:

6.

3. A recombinant nucleic acid encoding the bispecific antibody as described in claim 1.

4. A recombinant vector comprising the recombinant nucleic acid of claim 3.

5. A host cell comprising the recombinant nucleic acid of claim 3 or the recombinant vector of claim 4.

6. The use of the bispecific antibody of claim 1 in the preparation of a product for targeting infectious bursal disease virus and promoting the proliferation of chicken spleen lymphocytes.

7. A product that targets infectious bursal disease virus and promotes the proliferation of chicken spleen lymphocytes, comprising the bispecific antibody as described in claim 1.

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